DNA damage response is critically important for the maintenance of genomic integrity and tumor suppression. A key player involved in DNA damage response is MDC1, the Mediator of DNA damage Checkpoint protein 1. Over the years, we have shown that MDC1 acts downstream of H2AX phosphorylation and is involved in the regulation of many ATM-dependent events. More recently, we demonstrated that MDC1 also initiates a RNF8 and ubiquitination-dependent signaling pathway, which is required for the accumulation of many DNA damage checkpoint and repair proteins at sites of DNA breaks. We now know that the regulation of this ubiquitination-dependent cascade is quite dynamic, since an ubiquitin specific protease USP3 can counteract RNF8 function and remove ubiquitin moiety from histones. Such ubiquitination and deubiquitination cycle controlled by RNF8 and USP3 may be involved in DNA damage signal transduction. In addition, we have isolated TopBP1 as a new MDC1-associated protein. Since TopBP1 is mostly known for its role in replication stress pathway and its direct involved in the activation of ATR kinase, we reason that MDC1 may have a previously uncharacterized role in replication checkpoint control. It may stabilize TopBP1 at stalled replication forks, act to amply replication stress signals and thus enhance ATR activation and replication checkpoint control. Based on these preliminary studies, we propose to: 1) study MDC1 function in replication checkpoint control;2) investigate the roles of USP3 in DNA damage response;3) determine the functional significance of RNF8/USP3-dependent pathway in DNA damage repair and tumorigenesis in vivo. PUBLIC HEALTH RELEVANCE: MDC1 (Mediator of DNA damage checkpoint protein 1) is a key DNA damage checkpoint protein, which we identified and studied for several years. We now know that MDC1 controls multiple signaling pathways involved in DNA damage response. Further studies of MDC1 functions in DNA damage response will allow us to understand the complexity of DNA damage signaling pathways and how cells utilize multiple redundant and overlapping pathways to ensure genomic integrity.